Alzheimer's disease (“AD”) is a neurodegenerative disorder characterized by the occurrence of amyloid plaques, neurofibrillary tangles and significant neuronal loss. β-Amyloid protein (also referred to as the Aβ peptide), the main component of senile plaques, has been implicated in the pathogenesis of Alzheimer's disease (Selkoe (1989) Cell 58:611-612; Hardy (1997) Trends Neurosci. 20:154-159). β-Amyloid has been shown to be both directly toxic to cultured neurons (Lorenzo and Yankner (1996) Ann. NY Acad. Sci. 777:89-95) and indirectly toxic through various mediators (Koh et al. (1990) Brain Research 533:315-320; Mattson et al. (1992) J. Neurosciences 12:376-389). Additionally, in vivo models, including the PDAPP mouse and a rat model have linked β-amyloid to learning deficits, altered cognitive function, and inhibition of long-term hippocampal potentiation (Chen et al. (2000) Nature 408:975-985; Walsh et al. (2002) Nature 416:535-539). Therefore, a great deal of interest has focused on therapies that alter the levels of β-amyloid to potentially reduce the severity or even abrogate the disease itself.
One AD treatment strategy that has recently emerged in response to successful studies in PDAPP mouse and rat experimental models, is that of passive immunization of individuals to provide immunoglobulins such as antibodies specific to β-amyloid. (See e.g., Bard et al. (2000) Nat. Med. 6:916-919 and Bard et al. (2003) Proc. Natl. Acad. Sci. USA 100:2023-2028). Recently, it has also been shown that Abeta reduction by passive immunization protects against the progressive loss of synaptic degeneration in a transgenic mouse model of Alzheimer's disease. (Buttini et al. (2005) J. Neurosci. 25:9096-101).
Recent advances in recombinant technology have allowed for the production of antibodies against virtually any target, for example, cancer cells, bacteria, and viruses. Typically, an antibody is produced using a cell line that has been engineered to express the antibody at high levels. The engineered cell line is subsequently grown in a culture that comprises a complex mixture of sugars, amino acids, and growth factors, as well as various proteins, including for example, serum proteins. However, separation of complete antibodies from cell by-products and culture components to a purity sufficient for use in research or as therapeutics poses a formidable challenge. The purification of the antibody molecules is especially critical if the antibodies are to be used as a drug for administration to humans.
Traditional antibody purification schemes (or trains) often comprise a chromatography step which exploits an ability of the antibody molecule to preferentially bind or be retained by the solid phase (or functionalized solid phase) of a chromatography column compared to the binding or retention of various impurities. Schemes have been proposed or carried out to purify antibodies which first bind CH2/CH3 region-containing proteins to Protein A immobilized on a solid phase, followed by removal of impurities bound to the solid phase by washing the solid phase with a hydrophobic electrolyte solvent and the subsequent recovery of the CH2/CH3 region-containing proteins from the solid phase. However, these schemes are limited in that the conditions used to preferentially bind the CH2/CH3 region-containing proteins also support binding of impurities (e.g., antibodies with incomplete CH2/CH3 regions). In the development of human therapeutics, such impurities are highly undesirable.
Accordingly, a need exists for improvements in the purification of proteins or polypeptides having constant regions, in particular, proteins having Fc regions (e.g., antibodies), produced in cell culture.